The broad objective of this proposed research is to determine the mechanisms by which flavoproteins promote catalysis of oxidation-reduction and non-redox reactions. Specifically, the role of flavin in non-redox catalysis will be investigated by elucidating the reaction mechanism of D-hydroxynitrile lyase. Since this enzyme catalyzes a physiologically significant non-redox reaction but also exhibits properties similar to the flavoprotein oxidases, the relationship between the oxidase-like chemical reactivity of the prosthetic group and its ability to act as a catalyst in non-redox reactions will also be determined. Studies with pig liver glycolic acid oxidase will be conducted to determine the mechanism of hydrogen transfer (proton versus hydride), the participation of reduced flavin as the acceptor of substrate hydrogen and the role of flavin-substrate adducts as intermediates in oxidative catalysis by flavoproteins. The mechanism of action of these enzymes will be elucidated in studies involving: kinetic analysis, static and rapid reaction spectrophotometry, substrate analogues, flavin analogues containing modifications in the isoalloxazine nucleus and the N-10 side chain, radioactive tracers.